Reactive oxygen species accelerate de novo acquisition of antibiotic resistance in E. coli

Summary Reactive oxygen species (ROS) produced as a secondary effect of bactericidal antibiotics are hypothesized to play a role in killing bacteria. If correct, ROS may play a role in development of de novo resistance. Here we report that single-gene knockout strains with reduced ROS scavenging exhibited enhanced ROS accumulation and more rapid acquisition of resistance when exposed to sublethal levels of bactericidal antibiotics. Consistent with this observation, the ROS scavenger thiourea in the medium decelerated resistance development. Thiourea downregulated the transcriptional level of error-prone DNA polymerase and DNA glycosylase MutM, which counters the incorporation and accumulation of 8-hydroxy-2′-deoxyguanosine (8-HOdG) in the genome. The level of 8-HOdG significantly increased following incubation with bactericidal antibiotics but decreased after treatment with the ROS scavenger thiourea. These observations suggest that in E. coli sublethal levels of ROS stimulate de novo development of resistance, providing a mechanistic basis for hormetic responses induced by antibiotics.


INTRODUCTION
][6] Non-standard dosing regimens expose animals or environmental microbes to sublethal levels of antibiotics for prolonged periods of time. 7,8hese long-term sublethal levels of antimicrobials facilitate the selection of drug-resistant mutants, horizontal transfer of antimicrobial resistance genes, and de novo generation of drug resistance. 9,10Resistant variants of many microbial species can easily reach humans through multiple routes, including animal-derived food products, contaminated water, and soil, among others. 11,12ntibiotics such as quinolones, which specifically target DNA, have been observed to induce de novo DNA mutations partially through the activation of the SOS response. 13,14][17] However, it remains unclear whether exposure to other antibiotics that do not directly damage DNA induces mutations and facilitates development of drug resistance.
Reactive oxygen species (ROS) are hypothesized to function as a secondary killing mechanism when bacteria are exposed to certain bactericidal antibiotics, like aminoglycosides, quinolones, and b-lactams. 18The specific drug-target interactions of these antibiotics generally promote the oxidation of NADH through the tricarboxylic acid (TCA) cycle-dependent electron transport chain (ETC). 19][22] Bacteria generate mutations as a consequence of exposure to antimicrobial agents.These DNA mutations include mutations directly related to antibiotic resistance, mutations related to stress response, and mutations related to metabolism and other vital activities. 23ifferent antibiotics cause different mutations due to their particular drug targets.However, the specific formation mechanism of these mutations and whether they have a certain commonality are still unclear.ROS can cause DNA damage during cell replication, and subsequently mismatched or unpaired damaged DNA can cause the formation of mutations. 24,257][28][29] Whether ROS produced under long-term low-dose exposure to bactericidal antibiotic is a common mutagenic mechanism is still under debate.If yes, one of the possible mechanisms would be the induction of the SOS response to repair ROS-caused DNA damage, thereby increasing the probability of resistance formation.Based on these observations and considerations, we hypothesized that sublethal levels of ROS during bactericidal antibiotics exposure may either directly or indirectly cause mutagenesis, thereby stimulating ll OPEN ACCESS antibiotic resistance development.Thus, depending on the concentration, ROS could be both a beneficial and a killing agent for bacteria upon exposure to antibiotics, thus forming a mechanistic basis for the observed hormetic dose responses. 30n this study, we investigated whether the production of ROS by E. coli due to long-term exposure to sublethal levels of bactericidal antibiotics accelerates de novo resistance acquisition.The following questions were addressed.
(1) Does the reduction of intracellular ROS by the scavenger thiourea (TU) effectively reduce mutation rates and decelerate resistance development?(2) Do the single-gene knockout strains related to ROS removal have increased in intracellular ROS levels and accelerate the development of drug resistance?(3) Are there shared mutations in strains resistant to bactericidal antimicrobials and can any patterns be discerned in the mutations that accompany the development of resistance?(4) Are intracellular 8-HOdG levels and/or expression levels of genes involved in DNA repair associated with the rate of development of resistance?
The outcome suggests a shared mutagenic mechanism as a result of exposure to bactericidal antibiotics induced by ROS in accordance with the hormesis principle, accelerating the development of resistance.This in turn suggests potential usefulness of antioxidants as strategy to mitigate development of antibiotic resistance.

ROS scavenger TU reduced the rate of resistance development
To investigate the role of ROS on de novo acquisition of antimicrobial resistance, we exposed wild-type E. coli to step-increasing sublethal concentrations of four antibiotics in the absence and presence of the ROS scavenger TU and documented antibiotic resistance evolution.These experiments follow up on our studies on the formation of ROS as an effect of exposure to antibiotics 31 and development of resistance under anaerobic conditions. 32According to the ''radical-based'' theory assuming a common killing mechanism for bactericidal antimicrobials, bacteria generate ROS when exposed to bactericidal antibiotics such as amoxicillin, enrofloxacin, and kanamycin. 18,33In contrast, the bacteriostatic antibiotics exposure generates negligible ROS in cells, so we used tetracycline as a control.Superoxide and hydroxyl radicals are scavenged efficiently by TU.Hence, resistance development of TU-treated cultures was compared to the wild type.The TU-treated cultures had lower rates of resistance buildup against the bactericidal compounds than TU-absence cultures (Figures 1A-1C).During amoxicillin and kanamycin exposure, differences between the wild-type and TU-added cultures were observed from the early stage, as evidenced by significantly higher minimum inhibitory concentrations (MICs) in the wild type at day 10, day 20, and day 30 (Figures 1E and 1G).The most noticeable difference between the wild-type and TU-treated cultures during enrofloxacin exposure started from day 20, when the MIC of the wild-type-resistant strain became significantly higher than that of the TU-treated-resistant strain (Figures 1B and 1F).The final resistance concentrations were lower in one replicate of amoxicillin, enrofloxacin, and both replicates of kanamycin exposure in combination with TU treatment.In the case of the bacteriostatic tetracycline, the final concentration was much lower than the concentration after exposure to bactericidal antibiotics, and there were no meaningful differences between the TU-treated cells and the wild type in resistance acquisition rate and MIC (Figures 1D and 1H).

ROS produced during bactericidal antibiotic exposure and TU treatment
To ascertain the presence of ROS at the moment that the cell was exposed to increased levels of an antibiotic, the intracellular levels of ROS were visualized using widefield fluorescent microscopy.Cells were taken from cultures at the middle stages where the MIC increased rapidly when the cells reached the resistance concentrations of 32 mg/mL.Time-lapse images showed that the intracellular presence of ROS was low immediately at the start of the exposure to one of the bactericidal antibiotics, increased after 3-5 h, and decreased again approximately 2 h later (Figures 2A-2C).ROS production levels were highest at 6 h of amoxicillin or enrofloxacin exposure, or 4.5 h of kanamycin exposure.Even when taking the time dependence into account, the distribution over the population was not homogeneous in the amoxicillin and enrofloxacin incubations.Hardly any ROS was observed in the tetracycline-exposed strain (Figure 2D).Subsequently, ROS production was quantified by flow cytometry in these strains exposed to 32 mg/mL amoxicillin, enrofloxacin, kanamycin, and tetracycline (Figure 2E).The bactericidal antibiotics induced higher ROS levels than the bacteriostatic tetracycline.TU treatment significantly reduced the ROS levels caused by exposure to bactericidal antibiotics.Flow cytometry sorting was applied to divide the ROS positive and negative populations in these strains when ROS levels were highest, and the DNA of the separated populations was sequenced entirely.No different mutations between the ROS positive and negative populations were observed (Table S1).
ROS elimination-associated single-gene knockout strains acquire resistance faster While the previous set of experiments examined the effects of reduced ROS on the development of resistance, the effect of increased ROS was investigated using six single-gene knockout E. coli strains, DsodA, DsodB, DsoxR, DsoxS, DkatE, and DyggX that are deficient in ROS elimination.SodA and SodB are superoxide dismutase. 34SoxR activates the transcription of SoxS, and both SoxR and SoxS participate in the removal of superoxide. 35The KatE enzyme is the monofunctional catalase, which decomposes hydrogen peroxide into water and oxygen. 36YggX is a putative Fe 2+ -trafficking protein, which is proposed to play a role in the oxidation resistance of iron-sulfur clusters. 37We assume that when these genes are removed or disabled, bacteria will lose part of the ability to remove excess ROS, thus increasing the ROS levels during antibiotic treatment.
After prolonged amoxicillin exposure, maximum resistance concentrations of the mutants and wild-type were similar (Figure 3A).However, the rates of resistance development in these single-gene knockout strains were faster than those in the wild type during the middlelate stages, as evidenced by significantly higher MIC in DsodA, DsoxR, and DsoxS at day 20 (Figure 3E).For enrofloxacin, the MIC of the knockout strains started to increase after day 10.Both DsoxR and DyggX exhibited significantly higher MIC compared to the wild type at day 10 and day 20, while the MIC of DsodA and DkatE was also higher than that of the wild-type-resistant strain at day 20 (Figures 3B and  3F).The final enrofloxacin resistance of the knockout-mutant strains reached maximum concentrations of 2,048 mg/mL, double compared to the wild type (Figure 3B).Of all knockout-mutant strains except DsoxS only one replicate could reach the maximum concentration.The other replicates were killed while evolving to resist to high concentrations of the antibiotic, probably by the elevated ROS levels.Despite this, the surviving replicates in DsodA, DsoxR, DkatE, and DyggX showed faster resistance acquisition corroborating that ROS at these levels lead to cellular conditions that are at a tipping point between being beneficial and detrimental.During kanamycin exposure, all mutant strains reached the same maximum resistance concentrations as the wild type (Figure 3C).However, mutant strains reached 2,048 mg/mL faster than the wild type, and the MICs of DsodA and DsoxR were significantly higher than that of the wild type at day 10 (Figure 3G).No notable differences were observed in resistance development during tetracycline exposure; only DyggX had a slightly higher MIC than the wild-type and the other mutants.We doubt this is biologically meaningful, as the rate of resistance development was roughly the same as that of the wild type (Figures 3D and 3H).
ROS level elevated after knockout of sodA or soxR during exposure to bactericidal antibiotics Subsequently, we measured the ROS production levels under two conditions.Before inducing resistance, the naive strains were treated with one-quarter MIC antibiotics, and after acquiring de novo resistance, the final resistant strains were treated with the maximum resistance concentrations of these four antibiotics.We chose two single-gene knockout strains DsodA and DsoxR because these strains acquired resistance faster compared to the wild type during exposure to bactericidal antibiotics (Figure 3).In addition, the wild type and TU-treated wild type were subjected to the measurement as well.The antibiotic-induced ROS production levels were lower in naive wild-type strains compared to the de novo resistant strains (Figures 4A and 4B).TU treatment significantly decreased the ROS levels during amoxicillin and enrofloxacin exposure (Figure 4A).ROS production was significantly increased in DsodA during enrofloxacin and kanamycin exposure, and in DsoxR during enrofloxacin exposure (Figure 4A).Higher but still non-lethal ROS production was detected in final resistant mutant strains (Figure 4B).TU treatment decreased ROS levels, compared to the wild type.DsodA and DsoxR significantly increased the ROS production levels during exposure of the maximal bactericidal antibiotics (Figure 4B).The ROS production levels in the naive strains or the de novo resistant strains were low during tetracycline treatment (Figures 4A and 4B).In short, when ROS scavenger genes were knocked out, the rate of antibiotic resistance acquisition was higher, accompanied by increased ROS accumulation levels.Together with the TU-treated condition, we conclude that sublethal ROS increase in cells correlated enhanced antibiotic resistance development.

TU decreased the mutation rate during exposure to bactericidal antibiotics
Acquisition of antibiotic resistance is known to be accompanied by the accumulation of mutations. 38To examine whether ROS is related to mutation formation, we performed fluctuation assays to check the spontaneous mutation rate in different ROS-producing strains.The E. coli wild-type, TU-treated wild-type, DsodA, and DsoxR naive strains were treated with one-quarter MIC of the four antibiotics used throughout this study.50 mg/mL rifampicin LB plates were used to select the mutated cells.The mutation rate was significantly increased after enrofloxacin and kanamycin exposure in the wild-type and in DsoxR strains compared with the untreated control (Figure 5).Kanamycin exposure also significantly increased the mutation rate in DsodA.Compared with the wild type, TU treatment significantly decreased the mutation rate during exposure to the bactericidal antibiotics amoxicillin, enrofloxacin, and kanamycin.Tetracycline exposure did not influence the mutation rate.In summary, the spontaneous mutation rate was related to ROS production, and TU treatment decreased the mutation rate.
The main antimicrobial resistance mechanisms by which genes are mutated after amoxicillin exposure are antibiotic inactivation, target alteration, efflux pumps, and reduced permeability (Figure 6A).All amoxicillin-exposed strains contained mutations in ampC and ompC.AmpC is a b-lactamase with substrate specificity for amoxicillin.The mutations observed in ampC were located in the promoter area (Table S2).These mutations could trigger the ampC amplifications observed before. 40Each de novo resistant strain carried an ampC amplification contig, except one replicate in DkatE.These contigs had different lengths and copy numbers, but they all affected the degradation of amoxicillin (Table 1).OmpC is an outer membrane porin and mediates the entry of various antibiotics including b-lactams. 41Another frequent mutation is envZ, which is the sensor histidine kinase of the EnvZ/OmpR two-component system and regulates OmpF and OmpC to modulate osmosis. 42In DkatE and DyggX strains, an excision of prophage element e14 was found, which is known to occur upon SOS response activation. 43xposure to enrofloxacin induced the mutations in gyrA that are usually involved in de novo enrofloxacin resistance. 44Similar mutations were evolved in the genes coding for gyrase subunit gyrB, DNA topoisomerase 4 subunit parC, and parE (Figure 6B).The single-gene knockout strains accumulated mutations in HTH-type transcriptional regulator acrR, which regulates acrAB genes.In the wild type and the TU-treated wild type, mutations in acrA were observed, which codes for a multidrug efflux pump that uses the electrochemical proton gradient to export antibiotics. 45Several mutations were observed in genes associated with oxidative stress response, the ETC, SOS response, and DNA repair, especially in the single-gene knockout strains.This indicates that ROS is involved in resistance mutation acquisition.
The frequently mutated genes after kanamycin exposure are fusA, kdpD, sbmA, trkH, and atpG (Figure 6C).Mutations in atpG and op-pABCDF have not yet been recorded in the Comprehensive Antibiotic Resistance Database (CARD), even though these mutations emerged after aminoglycosides antibiotics exposure. 46,47Comparable to enrofloxacin-exposed strains, kanamycin-exposed strains developed mutations in genes that are involved in ETC, SOS response, and DNA repair.This further strengthens the notion that ROS is involved in the development of de novo drug resistance.
Tetracycline exposure caused the fewest mutations (Figure 6D).The mutations that occurred caused target alteration or appeared in genes involved in coding for efflux pumps.The genes mutated after tetracycline exposure were sometimes also mutated after exposure to other antimicrobials, albeit in different positions (Table S2).For instance, the gene coding for RNA polymerase subunit rpoBC was mutated also after amoxicillin treatment in wild-type E. coli and in the DsodA mutant (Figure 6A).The multidrug efflux pump subunit AcrA and regulator AcrR have been shared with enrofloxacin-resistant strains (Figure 6B).The mlaA gene that was mutated in most tetracycline-exposed strains is closely associated with ompC.''Radical-based'' theory suggests that ROS has a secondary role in cell killing by damaging DNA and nucleotide pools.However, cells have damage-repair mechanisms such as base excision repair and mismatch repair.Incorrect repair causes mutations in surviving bacteria, eventually promoting also beneficial mutations and thus resistance acquisition.To check transcription levels of DNA damage-repair-related genes, we performed RNA sequencing on cells exposed to antimicrobials.During amoxicillin exposure, the transcription of the DNA damage-inducible genes of the SOS response lexA, umuC, umuD, mutM, yebG, and nudG was upregulated.The increase in the transcription level was attenuated when TU was added (Figure 7A).Transcript levels of recA, recN, and recX were similarly upregulated; addition of TU made no difference.The up-or downregulation of genes related to DNA damage repair showed the greatest change during enrofloxacin exposure.Compared with the TU-treated group, only recF, uvrA, mutM, and vsr displayed clearly higher expression levels in the group without TU.Most of the other genes, like dinB, dinI, recA, recN, and recX, were upregulated to similar levels with or without TU addition.After kanamycin exposure, transcription levels of umuC, umuD, and mutM in the wild type were higher than those in the TU-treated group.Treatment with the bacteriostatic tetracycline showed the lowest transcription levels, and no clear difference due to the addition or absence of TU.Interestingly, the gene whose transcription level was always upregulated after bactericidal antibiotic treatment and whose transcription level was higher in the absence of TU was mutM.MutM is involved in base excision repair of DNA damaged by the mutagenic lesion 8-HOdG, and other oxidized nucleobase damage. 49On the one hand, the upregulation of base excision repair gene mutM in the TU-absence group indicates that cells faced more oxidative stress during bactericidal drugs exposure, which suggests a higher mutagenesis frequency compared to the TU-treated cells.On the other hand, TU treatment decreased the mutagenesis caused by oxidative damage thus presumably slowing down mutagenesis and, also, lowering the changes of acquiring beneficial stress-related mutations.
To further clarify the emergence of the DNA damage biomolecule 8-HOdG, we measured its levels after antibiotic treatment.We choose the same strains as for RNA sequencing and performed the same treatment.In the TU-absence group, the 8-HOdG levels were significantly increased after bactericidal antibiotics exposure (Figure 7B).Only enrofloxacin treatment elevated the 8-HOdG levels in the TU-treated culture.Compared with the TU-absence strains, the 8-HOdG levels in the TU-treatment culture were significantly reduced.Together with the transcriptomics, we conclude that the ROS-induced DNA mutagenesis agent 8-HOdG was increased during the bactericidal antibiotics' exposure and decreased after TU treatment.

DISCUSSION
The central question this study addressed is whether the ROS formed in E. coli upon exposure to bactericidal antibiotics affects development of resistance according to the principle of hormesis (Figure 8).In this particular case, low levels of ROS-induced stress would stimulate adaptation to the presence of bactericidal antibiotics, while high levels damage the cell or kill it.This hypothesis was postulated based on the capacity of ROS to induce DNA damage, such as the formation of 8-HOdG, which triggers the SOS response and activates the low-fidelity errorprone DNA repair systems, ultimately leading to increased mutations accumulation. 50,51Two main observations support this notion: 1) mutants that have reduced capabilities to eliminate ROS have higher rates of resistance development and 2) removal of ROS by TU decreased rates of resistance development.This suggests a mechanism for ROS-induced hormesis: killing occurs during short-time, high-dose antibiotic exposure, while the mutagenic effect is exerted during long-term, non-lethal dose exposure. 32This effect was more noticeable when E. coli was exposed to amoxicillin and kanamycin than in the case of enrofloxacin, most likely because the first two increased metabolic rates more than enrofloxacin.The increased metabolic rates cause the increase in ROS, as demonstrated by the low ROS levels in tetracycline-exposed cells, which are known to have lower metabolic rates. 52OS can cause over one hundred different 2-deoxyribose modifications and oxidative nucleobase lesions. 26The electrophilic hydroxyl radical ($OH) can directly react with DNA nucleobases.For example, hydroxyl radicals attack the C5 = C6 double bonds of thymine or cytosine. 53Fragmented formamidopyrimidine is formed by the hydroxyl radical-induced imidazole ring opening in guanine and adenine. 54Additionally, 8-HOdG is generated from hydroxylation of guanine in nucleotide pools or the genomic DNA level. 55Instead of pairing with cytosine, In the table the size of the fragment, the amplification factor, and upstream and downstream of the genes contained in the fragment are listed for each strain tested.
8-HOdG prefers to pair with adenine thus causing frequent guanine-to-thymine mutations during replication, 29 which were also observed in our experimental system. 32Consistent with this notion, the 8-HOdG level was increased after bactericidal antibiotics exposure, and it was significantly reduced after the application of ROS scavenger TU.These findings suggest that the emergence of ROS creates favorable conditions for elevated mutation rates.In addition to this direct nucleobase damage, hydroxyl radicals can interact with lipids, leading to the formation of malondialdehyde and 4-hydroxynonenal. 56These lipid peroxidation products can react with adenine, guanine, and cytosine to form mutagenic adducts. 57Along with the nucleobase damage, ROS can also compromise single-strand break and double-strand break. 27,28n response to ROS-induced DNA damage, bacteria activate SOS-response genes, firstly lexA and recA. 58RecA searches for damaged DNA, while the breakdown of LexA activates SOS genes involved in damage repair. 59The activation of repair systems can exhibit a general nature or be specific to various antibiotics.During exposure to several antibiotics, the transcription levels of recA and lexA did not show an obvious common pattern between TU-treated or untreated conditions (Figure 7A).However, other damage-repair genes which are subsequently activated by the SOS response were upregulated by bactericidal antibiotic treatment, and this upregulation was attenuated by the addition of TU, for example, umuC and umuD upon amoxicillin or kanamycin exposure and recF and uvrA after enrofloxacin exposure.This indicates that transcription levels of DNA damage-repair genes differ as a result of exposure to different antibiotics and possibly of different ROS levels.Exposure to enrofloxacin yielded more upregulated genes, conceivably because enrofloxacin targets DNA gyrase and hence causes more direct DNA damage.Transcription levels of mutM were all upregulated upon exposure to the three bactericidal antibiotics and attenuated by TU treatment.mutM codes for a formamidopyrimidine-DNA glycosylase, with an inclination for, but not limited to, base excision repair for 8-HOdG.For example, it also recognizes and removes oxidized purines, and some oxidized pyrimidines, to leave an apurinic or apyrimidinic (AP) site. 60,61The transcription regulation of this glycosylase MutM characterizes the DNA damage-repair process that is activated upon exposure to bactericidal antibiotics.SOS response-induced genes, such as umuC and umuD which code for error-prone DNA polymerase V, contribute to an increase of mutation rates. 62,63][66][67] The low-fidelity polymerase V is derepressed when DNA is damaged.Due to the absence of an intrinsic 3 0 -5 0 exonuclease proofreading activity, it is unable to rectify errors during translesion DNA synthesis. 68This error, however, creates the potential to overcome the DNA damage caused by ROS, thus producing damage-inducing mutations in the E. coli genome. 69During long-term sublethal levels of antibiotic exposure, mutations that favor resistance development will be selected from these damage-inducing mutations.This offers an opportunity for research on drugs that target the SOS response and so aim to reduce the development of drug resistance.For instance, N 6 -(1-naphthyl)-ADP prevents the formation of the RecAÀDNA filament. 70Suramin inhibits the RecA-induced cleavage of LexA. 71Antioxidant drugs such as N-acetylcysteine can attenuate ROS levels, SOS response induction, and mutagenesis during ciprofloxacin treatment. 72gure 8. Scheme depicting the chain of events by which non-lethal concentrations of bactericidal antimicrobials cause mutations resulting in resistance to the specific antibiotic In brief: TCA cycle activity mediated oxidation of NADH may lead to the emergence of superoxide causing damage to iron-sulfur cluster containing proteins which with hydrogen peroxide present can lead to the generation of the hydroxyl radical.The ensuing genome damage can promote the SOS response which, if initial levels of the reactive oxygen species (ROS) are sublethal, may promote mutation generation in surviving cells some of which are beneficial for antibiotic survival.The data presented are summarized by the processes and enzymes indicated and provide a mechanistic basis for the observed hormetic dose responses induced by antibiotics in bacteria. 30he various bacterial strategies for resistance development are reflected in the whole-genome sequencing results.In general, these consist of pumping out, reducing entry, or degrading the drugs and of protecting, modifying, or changing the expression of drug targets. 73Although the strains at the end of each antibiotic treatment had evolved in a different manner, the final results showed commonality.The most characteristic mutations are in b-lactamase after amoxicillin treatment, DNA topoisomerase after enrofloxacin treatment, and elongation factor G after kanamycin treatment.E. coli increases the expression of b-lactamase by amplifying ampC, which is also evidenced by the fragments we observed containing ampC amplification. 74These contigs could come in different sizes, had copy numbers between 20 and 600, and were horizontally transferred, thereby making a sensitive E. coli receptor strain highly amoxicillin resistant. 40,75Quinolone-induced mutations are often clustered within small regions of DNA topoisomerase-coding genes, known as quinolone resistance-determining regions. 76Mutations may lead to structural changes in the target site of the enzyme tetramer, resulting in reduced affinity for quinolones and thus resistance to these drugs. 77When E. coli is exposed to enrofloxacin, gyrA was first mutated, followed by gyrB, parC, or parE. 44Elongation factor G (EF-G) fusA catalyzes the guanosine triphosphate (GTP)-dependent ribosomal translocation during translation elongation. 78Mutations in this gene may prevent kanamycin from binding EF-G and preventing translation.0][81] In addition to these target-specific mutations, other mutations, such as reduced membrane permeability and efflux pumps, appeared in combination.In this study, we also found mutations in many genes related to oxidative stress and SOS response.This is additional evidence that ROS and SOS response are involved in the evolution of drug resistance.Moreover, this explains why strains that had developed resistance to bactericidal antibiotics and were next exposed to other bactericidal antibiotics showed increased rates of resistance acquisition. 23ROS and the subsequently activated SOS response accelerate resistance evolution in E. coli exposed to stepwise-increasing sublethal levels of bactericidal antibiotics.

Limitations of the study
This study demonstrates the role of ROS in the development of antimicrobial resistance.Development of resistance in E. coli exposed to stepwise-increasing sublethal levels of bactericidal antibiotics was slowed down by treatment with the antioxidant TU, and mutants with reduced ROS scavenging showed accelerated resistance acquisition.However, no significant increase in the mutation rate was found in the mutant strains compared to the wild type upon bactericidal antibiotics exposure which may well reflect that we have yet to pinpoint the exact concentration of ROS that is responsible for the tipping point from ROS-induced cell death to ROS-induced beneficial mutation acquisition.In future, we may strengthen the verification of our results by knocking out multiple genes or using the ROS-inducing chemicals such as hydroxyurea to come closer to the indicated concentration domain. 82Considering that the purpose of our research is to understand the formation of resistance and try to reduce or slow down the resistance acquisition, our study demonstrates the feasibility of using antioxidants to reduce excess ROS formation in vivo and in that manner reduce drug resistance development by inhibiting SOS responses.''as TU-added'' 100mM thiourea was added to the medium.Cultures were incubated in tubes with a start optical density (OD) 600nm of 0.1 at 37 C under constant shaking at 200 rpm.The kanamycin cassette in Keio strains was replaced by temperature-sensitive pCP20 plasmid.

METHOD DETAILS Evolution experiments
The evolution experiments aimed at inducing resistance were conducted in accordance with the previously established methodology. 95riefly, susceptible naive E. coli K12 strains of wild-type, TU-treated wild-type, and single-gene knockout strains (DsodA, DsodB, DsoxR, DsoxS, DkatE, DyggX) were exposed to one-quarter MIC of amoxicillin, enrofloxacin, kanamycin, or tetracycline.An untreated control group without antibiotics was also included.After 24 h of incubation, if the OD 600 of the antibiotic-exposed culture was equal to or greater than 75% of the OD 600 of the antibiotic-free culture, the antibiotic-exposed cells were selected and inoculated with double concentrations of the respective antibiotic, while retaining the previous low concentrations as a back-up.After another 24 h of incubation, if the OD 600 of the high antibiotic concentration culture was equal to or greater than 75% of the OD 600 of the low antibiotic concentration culture, cells from the high concentration culture were used; otherwise, cells from the low concentration culture were used, and the antibiotic concentration was doubled again.The experiment was stopped when the bacteria could no longer tolerate the higher antibiotic concentration.Each strain's resistance evolution was independently performed at least twice, and the antibiotic-free control groups were cultured in the absence of antibiotics but otherwise under identical conditions until the end of the experiment.
To monitor the development of resistance, MIC measurements were conducted three times a week.Cells were cultured in 96-well plates using a spectrophotometer plate reader (Thermo Fisher Scientific) with antibiotic concentrations ranging from 0.25 mg/mL to 2048 mg/mL, doubling at each step.The initial OD was 0.05 and the MIC was determined as the lowest concentration that resulted in a final OD 600 of less than 0.2.

ROS measurements
Fluorescence microscopy was performed to monitor ROS generation.The fluorescent dye 3 0 -(p-Hydroxyphenyl) Fluorescein (HPF) was utilized for the detection of ROS.Cells were sampled from cultures during the mid-stages of resistance development, specifically when they reached resistance concentrations of 32 mg/mL for each antibiotic.In each culture, HPF dye was added to achieve a final concentration of 10 mM, followed by incubation in a shaking incubator at 37 C for 40 min.Subsequently, bacterial cultures were centrifuged at 6000 rpm for 5 min, and the resulting pellet was thoroughly resuspended in medium.The samples were then treated with the corresponding antibiotic concentration, and 1.3 mL of each sample was pipetted onto a microscope slide glass with 2% agarose mixed with medium.Fluorescence detection was performed using the Nikon Eclipse Ti microscope equipped with NIS-elements AR software, employing an excitation/emission wavelength of 490/515 nm.The assembly of the time-lapse pictures was done using ImageJ software.
Flow cytometry (FACS) was used to quantify the level of ROS production.Overnight cultures were diluted to an OD 600 of 0.1 in fresh medium, followed by treatment with the corresponding antibiotics.The cultures were then incubated for 4 h at 37 C in a shaking incubator.Subsequently, a final concentration of 10 mM of the fluorescent dye HPF was added to each culture and incubated for an additional 40 min.All samples were centrifuged at 6000rpm for 5 min and the pellet was resuspended in 1 mL Evans medium without glucose.For the FACS sorting, BD FACSAria III Sorter was used with BD FACSDiva Software version 8.0.1.The laser settings were set on 250V FSC, 400V SSC, and 450V FITC (GFP).A total of 10,000 events were measured for each sample.The percentage of the ROS-positive population was determined, and each sample was independently replicated three times.

Mutation rate measurements
Fluctuation assays were performed to determine the mutation rate, rifampicin was used to select for gain-of-function mutations in the rpoB gene. 72The experimental procedure was carried out as follows: Susceptible E. coli K12 strains, including wild-type, TU-treated wild-type, DsodA, and DsoxR were exposed to one-quarter MIC of amoxicillin, enrofloxacin, kanamycin, and tetracycline.Each culture was incubated until reaching OD 600 of approximately 0.5-0.6.Next, 200 mL of each culture was plated onto LB agar plates containing rifampicin (50 mg/mL), while 100 mL of each culture was stepwise diluted to 10 À7 using medium and plated onto antibiotic-free LB agar plates.The plates were then incubated at 37 C for 24 h for the antibiotic-free LB plates and 48 h for the LB plates containing rifampicin.Colony-forming units (CFUs) were counted for both plate types.Mutation rates were calculated by Lea-Coulson Ɛ < 1.0, the comparison was done by the maximum likelihood ratio statistical test in webSalvador 0.1, which was powered by rSalvador 1.8. 39WebSalvador provided the maximum likelihood ratio value, along with the corresponding P-value and the upper and lower limits of the 95% confidence intervals, which were used to generate the bar graph.Statistical significance was indicated as *p < 0.05 and **p < 0.01.The experiment was independently replicated at least 10 times to ensure the reliability and reproducibility of the results.

Whole genome sequencing
The genomic DNA was extracted from the final resistant strains and the corresponding antibiotic-free strains utilizing the DNeasy Blood and Tissue Kit (Qiagen).Libraries were constructed using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England BioLabs) in combination with NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs; New England BioLabs), following the manufacturers' recommended protocols.Subsequently, the genomic DNA libraries were subjected to paired-end sequencing (2 x 150 bp) on the NextSeq 550 next-generation sequencing system (Illumina).The quality of the raw reads was assessed using FastQC and MultiQC.Adapter sequences were removed using Cutadapt.After removing the low-quality bases and optical duplicates, the reads were aligned to reference genomes of wild-type strains (NC000913) and Keio strains (CP009273) using Bowtie2, and the PCR duplicates were removed.Variant calling was performed using Freebayes and Lofreq, and variant annotation was conducted using Snpeff.Subsequently, the single nucleotide polymorphisms (SNPs) and small indels were inspected using the Integrative Genomics Viewer (IGV).The mutated genes in the resistant strains were compared to their corresponding control (antibiotic-free) strains, and shared mutated genes were excluded from further interpretation.In addition to analyzing small genomic alterations, copy number analysis by cn.MOPS was performed to identify larger genomic alterations, such as amplifications and deletions.

RNA sequencing
Total RNA was isolated and purified from the final stable resistant strains and their corresponding antibiotic-free strains using the RNeasy Protect Bacteria Kit (Qiagen).For RNA-Seq analysis, libraries were constructed following the manufacturer's protocols, employing the NEBNext rRNA Depletion Kit (Bacteria) (New England BioLabs) in combination with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Unique Dual Index Primer Pairs) (New England BioLabs).The resulting libraries were subjected to sequencing on a NextSeq 550 Sequencing System (Illumina) with read lengths of 75 bp.To ensure data quality, FastQC and MultiQC were employed for comprehensive quality control of the raw sequencing data.Subsequently, the reads underwent trimming procedures, and were aligned to the reference genomes (NC000913) using Bowtie2.For the determination of differential gene expression, normalized gene expression values were computed, and Log 2 fold changes were calculated by comparing the resistant strains to the antibiotic-free control by HTSeq and DESeq2.Within each treatment group, DNA damage-repair genes surpassing a cutoff of 2 were selected and incorporated into the generated heatmap.

8-HOdG level measurements
The DNA Damage Competitive ELISA Kit was employed to quantify the levels of the oxidative stress marker 8-hydroxy-2 0 -deoxyguanosine (8-HOdG) according to the manufacturer's instructions.In brief, the final stable resistant strains of wild-type and TU-treated wild-type were selected and subjected to treatment with the corresponding antibiotics.The cultures were incubated for 4 h at 37 C in a shaking incubator.Subsequently, the samples were centrifuged at 6000rpm for 5 min, and the resulting pellet was resuspended in 0.3 mL of Lysis Buffer (containing 10 mM Tris-HCl, 2 mM EDTA, 1% SDS).A 5-fold dilution of the samples was loaded onto the antibody-coated 96-well plate.Following the recommended protocol for incubation, the absorbance at 450 nm was measured using a spectrophotometer plate reader (Thermo Fisher Scientific).The concentrations of 8-HOdG in the samples were determined by referencing to a standard curve.Furthermore, the DNA concentrations of the samples were measured using a microvolume spectrophotometer (DeNovix) and utilized for normalization.As controls, antibiotic-free samples of wild-type and TU-treated wild-type were included, and each sample was independently replicated three times.

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses were conducted using IBM SPSS Statistics software.Detailed information regarding the statistical methods employed for each experiment can be found in the figure legends and corresponding figures.

Figure 1 .
Figure 1.ROS scavenger TU slowed down bactericidal antibiotics resistance development (A-D) Acquisition of resistance to amoxicillin (A), enrofloxacin (B), kanamycin (C), and tetracycline (D) of E. coli wild-type MG1655 (black lines) and the TU-treated (100mM) E. coli wild-type MG1655 (blue lines) strains.The x axis represents the duration of evolution in days, while the y axis represents the concentration of acquired resistance.(E-H) The minimum inhibitory concentration (MIC) at day 10, day 20, and day 30 for each resistant strain (WT and TU-treated E. coli) with respect to amoxicillin (E), enrofloxacin (F), kanamycin (G), and tetracycline (H) during the process of antibiotic resistance acquisition.Data are presented as means G SD, statistical significance was determined using a one-way ANOVA, N R 3, *p < 0.05, **p < 0.01.

Figure 2 .
Figure 2. ROS produced during bactericidal antibiotic exposure and attenuated by TU treatment (A-D) Time-lapse photography capturing the ROS generation in E. coli exposed to antibiotics treatment with cells resistant to 32 mg/mL amoxicillin (A), enrofloxacin (B), kanamycin (C), and tetracycline (D).The scale bar refers to 10 mm.(E) ROS production levels were quantified by flow cytometry.E. coli and TU-treated E. coli resistant strains were exposed to 32 mg/mL of amoxicillin (AMO), enrofloxacin (ENR), kanamycin (KAN), and tetracycline (TET), separately.The strains used are the same as A-D.Data are represented as means G SD, statistical significance was investigated using a one-way ANOVA, N = 3, **p < 0.01, ***p < 0.001.

Figure 3 .
Figure 3. ROS elimination-associated single-gene knockout strains gain antibiotic resistance faster (A-D) Acquisition of resistance to amoxicillin (A), enrofloxacin (B), kanamycin (C), and tetracycline (D) of E. coli wild-type MG1655 (black lines) and the singlegene knockout strains DsodA, DsodB, DsoxR, DsoxS, DkatE, and DyggX (colored lines).The x axis represents the duration of evolution in days, while the y axis represents the concentration of acquired resistance.(E-H) The MIC at day 10, day 20, and day 30 for each resistant strain (WT, DsodA, DsodB, DsoxR, DsoxS, DkatE, and DyggX E. coli) with respect to amoxicillin (E), enrofloxacin (F), kanamycin (G), and tetracycline (H) during the process of antibiotic resistance acquisition.Data are presented as means G SD, statistical significance was determined using a one-way ANOVA, N R 3, *p < 0.05, **p < 0.01.

Figure 4 .
Figure 4. ROS level elevated after knockout of sodA or soxR during exposure to bactericidal antibiotics (A) ROS production levels quantified by flow cytometry in naive wild-type strain, TU-treated strain, single-gene knockout strains DsodA and DsoxR, exposed to one-quarter MIC of amoxicillin, enrofloxacin, kanamycin, and tetracycline.Data are presented as means G SD, statistical significance was determined using a one-way ANOVA, N = 3, *p < 0.05, **p < 0.01 ***p < 0.001.(B) ROS production levels quantified by flow cytometry.The final resistant strains after resistance evolution, WT-resistant strain, TU-resistant strain, single-gene knockout-resistant strains DsodA and DsoxR, exposed to the maximum concentrations of amoxicillin, enrofloxacin, kanamycin, and tetracycline.Data are presented as means G SD, statistical significance was determined using a one-way ANOVA, N = 3, *p < 0.05, **p < 0.01 ***p < 0.001.

Figure 5 .
Figure 5.Effect of TU on the mutation rate during exposure to antibiotics Mutation rates were measured by fluctuation analysis.The naive strains of wild-type (WT), TU-treated wild-type (TU), DsodA and DsoxR, exposed to one-quarter MIC of amoxicillin (AMO), enrofloxacin (ENR), kanamycin (KAN), and tetracycline (TET).Cells were plated on rifampicin (50 mg/mL) LB plates for counting mutated cells, and LB plates for counting the total cell number.The CFU was counted after 48 h and analyzed by webSlvador.39Mutation rates were calculated by Lea-Coulson Ɛ < 1.0; the comparison was done by the maximum likelihood ratio statistical test.Error bar represented the upper and lower limit of the 95% confidence intervals, *p < 0.05, **p < 0.01, N = 10, CON = untreated control culture.

Figure 6 .
Figure 6.Mutated genes involved in antibiotic resistance, oxidative stress, and SOS response (A-D) The genomic DNA sequencing of each final resistant strain compared to each no-antibiotic-treated strain to identify mutated genes.The mutated genes are determined as defined by the Comprehensive Antibiotic Resistance Database (CARD).Rows show genes that observed mutations, and columns indicate different strains.Genes are grouped by functions.Red color indicates mutations observed in replicate one, green color mutations found in another replicate, purple color means mutations observed in both replicates, and black background indicates an absence of mutations.(A) amoxicillin exposure, (B) enrofloxacin exposure, (C) kanamycin exposure, (D) tetracycline exposure.

Table 1 .
Prolonged exposure to stepwise-increasing concentrations of amoxicillin causes the amplification of a chromosomal DNA fragment of varying length, but always containing ampC